The effect of titanium-implant surfaces on the behaviour and characteristics of osteoblasts
One of the strongest predictors of the clinical success of orthopedic and dental implants is the osteointegration of the implant to the damaged bone [1,2], a factor influenced also by the chemistry and topography of the surface of the implant [3-5]. These characteristics are critical for the development of appropriate bone formation on the healing area surrounding the implant.
After implantation, the surface of the implant is in contact with body fluids and tissues and thereby interacts with various cells, the most important of which are osteoblasts. Osteoblasts produce the extracellular matrix and assist in building bone, and are a major consideration in implant design; one of the main challenges in designing bone implants is efficiently attracting and activating osteoblasts. In order to produce mineralized bone, the osteoblasts must first adhere to the surface. Studies have shown that the adhesion, proliferation and differentiation of osteoblasts are related to the energy and roughness of the surface [6,7]; however, despite the vast amount of research concerning the field of implant surface roughness and its influence on osteoblasts, the optimal surface properties for the ideal adhesion and activation of osteoblasts have yet to be determined. At this time, most oral implants have moderate surface roughness, between 1.0 to 2.0 µm . Rough surfaces encourage entrapment of proteins, such as fibrin, and improve the mechanical properties of the implants [8-10].
Roughened implant surfaces can be created via a number of methods, most commonly by aluminum or titanium oxide grit-blasting followed by acid-etching . This method, however, may be problematic because it may lead to contamination of the surface by aluminum or titanium particles, which can result in inflammatory reactions and impaired bone formation [12,13]. Another method roughens the surfaces of titanium implants by using biocompatible and reabsorbable blasting materials, for example biphasic calcium phosphate (BCP) ceramic particles . Calcium-phosphate-based materials are sensitive to acid, making them easy to remove after the blasting process [15,16]. Moreover, animal experiments examining torque force and bone formation demonstrated a positive influence of BCP grid-blasting of implant surfaces, which can be attributed to the nano-scale topography created by this blasting method, which seems to increase the absorption of proteins and adhesion of osteoblasts [17-20]. This method results in random topography and a variety of chemical compositions, so the optimal properties of titanium implant surfaces are yet to be established .
In 2007, Le Guehennec et. al  compared the behavior of osteoblasts on various titanium surfaces. Four different groups were investigated: mirror-polished (Smooth-Ti), alumina-blasted and acid-etched (Alumina-Ti), sand-blasted, large-grit, acid-etched (SLA) and biphasic calcium phosphate grit-blasted and acid etched (BCP-Ti). Figure 1  displays the surfaces of the 4 investigated implants. Smooth-Ti shows parallel slits that are the result of the manufacturing process (Fig. 1-A), while the other three grid-blasted surfaces displays a rougher topography. In addition, residual alumina particles can be observed, embedded in the surface. The SLA implant showed a complex surface topography (Fig. 1-C), and the BCP-Ti surface presented irregular surface morphology. No residual particles could be found on the BCP-Ti implant after the acid-etching and cleaning, and it also had the roughest surface (data not shown).
Figure 1: Scanning electron microscope (SEM) analysis of the surfaces of the four implants: A – Smooth-Ti, B – Alumina-Ti, C – SLA, D – BCP-Ti. The white arrow in B points a residual alumina particle. Magnification X 100. Bar = 100 µm.
Table 1  shows the quantification of different elements that were left embedded on the surface of the implants after all the stages of processing.
Table 1: Semi-quantitative XPS analysis of the surfaces of the 4 different implant types.
Since contamination of the implant surface is a major concern, one of the main disadvantages of using alumina-blasted surfaces – the high Al content – is clear. But another element that hinders bone formation is carbon (C). These results illustrate that the presence of carbon in the BCP-grid implant is lower than the amount on SLA surfaces; therefore, BCP implants are more likely to undergo successful integration with the osteoblasts forming the bone. An explanation for this is likely due to the blasting and cleaning method of the SLA implant: the carbon, which contaminates the surface due to the blasting process, is buried deep in the grooves of the surface, decreasing the efficiency of cleaning procedures. In comparison, the method of BCP grit-blasting seems to result in less carbon contamination, so the implant contains fewer carbon molecules that might negatively effect bone formation.
Figure 2  presents the SEM micrographs of the osteoblast cell-line MC3T3-R1 after 2 days of culturing on the different surfaces.
Figure 2: SEM micrographs of the MC3T3-E1 cells grown on the different surfaces for 2 days. A – Smooth-Ti, B – Alumina-Ti, C – SLA and D – BCP-Ti. Magnification X 1000. Bar = 10µm.
All surfaces were covered with a uniform layer of osteoblasts, even though they spread less on the rough titanium surfaces compared with the polished surface. Cells grown on SLA surface developed more cytoplasmic extensions compared to Alumina-Ti and BCP-Ti.
The differentiation of the MC3T3-E1 cells grown on the different titanium surfaces and plastic surface (control) was assessed by alkaline phosphatase (ALP) activity assay (Figure 3) , ALP being an early marker for osteoblasts differentiation. ALP activity increased with time, as expected, and reached its highest levels after 15 days. After eight days of growing on the surfaces, no statistical difference was found between the groups, though cells grown on SLA and BCP-Ti displayed slightly higher ALP levels, and Smooth-Ti and Alumina-Ti showed slightly lower levels of ALP when compared to cells grown on plastic surface. After 15 days, however, significant differences were observed between Smooth-Ti and SLA, Alumina-Ti and SLA and SLA and BCP-Ti. After 21 days, no statistical differences were found between the groups.
This study demonstrates that the roughness, surface processing technique, and blasting material have a significant effect on the adhesion and differentiation of osteoblasts. Rougher surfaces seem to contribute to the differentiation of the osteoblasts, and the contamination by the blasting particles, such as aluminum and carbon particles, also appear to affect the osteoblasts differentiation and adhesion. Therefore, the BCP-grit blasting method, which combines the advantages of rougher implant surface and less contamination by alumina and carbon, seem to exhibit the best combination of surface topography and chemistry out of all the other implants investigated.
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